Validation of test system

To isolate human hERα variants with altered transcriptional activation activity (towards synthetic non-steroidal estrogen analogs), we utilized two efficient and sensitive high throughput screening methods based on macromolecular interaction in yeast. The first system was based on a modified yeast one hybrid screening which could be used for lacZ reporter gene assay and complemented phenotypic selection of library variants in vivo.

The second system was a very sensitive two hybrid assay for chemical complementation in yeast and thus could be used for high throughput ligand titration assay as well as for screening of large libraries.

3.1.1 Yeast one hybrid system:

To allow efficient identification of mutants of hERα LBD having an altered response to E2 as well as synthetic ligands, we used a modified yeast one hybrid system where a chimeric protein was generated consisting of Gal4 DNA binding domain and VP16 activation domain placed in the amino- and carboxylic- terminal end of ER LBD (GLV). This chimeric construct was introduced into yeast strain PJ69 4A, where HIS3 and ADE2 genes are driven by Gal1 and Gal2 promoter, respectively. Upon binding of ligand, the chimeric protein would be released from hsp chaperon, dimerize, transport inside the nucleus and bind to the different Gal promoters, thus allow the yeast cell to biosynthesize histidine (His) and adenine (Ade) in a media lacking His and Ade. This chemical complementation allowed us first to validate E2-LBD binding and later to look for orthogonal combination of ligand-mutant receptors in vivo on transformation plates. The yeast strain PJ69 4A has also β-galactosidase gene (lacZ) integrated under the control of another Gal promoter, Gal7. In presence of a functional ligand-receptor interaction, expression of lacZ would take place and a quantitative β-galactosidase assay could be done to reveal the quantity of induction, which was correlated to the binding of ligand to

nutritional selection and β-galactosidase assays are mentiond separately below:

3.1.1.1 On plate assay

The in vivo validation was done in yeast strain PJ69 4A by transforming with pGBT-LBD expressing the chimeric GLV or with the empty vector pGBT and plating the transformation mixture on minimal plates lacking adenine, histidine and tryptophan (SD-Ade-His-Trp). These plates were termed transactivation plate because of nutritional selection via inclusion of HIS3 and ADE2 genes as transcription reporter. These plates were supplemented with two different concentrations (0.1 and 1µM) of E2 or with EtOH as solvent control.

Transformation mix was also plated on SD-Trp plates for the selection of plasmid, elucidation of transformation efficiency and production of transformants for β-galactosidase assay. As shown in the figure 3.1, in the transactivation plates containing 0.1 or 1µM E2, cells harbouring LBD could grow whereas there was no growth on plates where cells were only harbouring the empty plasmid. There was no growth in the absence of ligand on transactivation plates, showing the stringency of the plate assay.

Figure 3.1: Ligand-dependent chemical complementation based on plate growth assay in yeast one hybrid system. Yeast strain PJ69 4A was transformed with plasmid pGBT (empty plasmid) or pGBT-LBD (chimeric construct Gal4 DBD- hERα LBD- VP16 AD) and the transformation mixture was plated on transactivation media (SD-Ade-His-Trp) with E2 or with solvent only. Growth was only observed with plates containing E2 where cells harbouring pGBT-LBD were plated.

As Gal7-lacZ was introduced in the strain PJ69 4A, the lacZ gene product ß-galactosidase could be used also as quantitative transcription reporter. In presence of β-galactosidase, the substarte o-nitrophenyl-β-D-galacto-pyranoside (ONPG) was hydrolyzed into galactose and o-nitrophenol, which is yellow in color and measured in a spectrophotometer at 420nm. We tested E2 and ligands CV2807, CV5407 and CV6019 at 10µM concentration. β galactosidase activity (Miller Unit) for E2 was considered to be 100% and relative activities were calculated for the above-mentioned ligands from the ß-galactosidase activity (Figure 3.2). When only the solvent was used instead of any ligand, the activity was negligible.

0 20 40 60 80 100 120

Estradiol CV2807

CV5407 CV6018

CV6019 EtOH

Relative Activity (%)

Figure 3.2: ß-galactosidase reporter gene assay in yeast one hybrid system, where PJ69 4A containing pGBT-LBD was inoculated with different ligands of 10µM concentration.

Ligand-induced transcriptional activation of lacZ was proportional to the β-galactosidase mediated breakdown of substrate ONPG into colored product o-nitrophenol, which is measured at OD420 nm and plotted against corresponding ligands in the graph to get the activity in Miller Unit. Taking E2 as 100%, the comparative activity was calculated for other ligands and plotted in the graph.

In the two hybrid system, the cDNA encoding hERα amino acids 312-595 containing most of the LBD domain (hERα amino acids 302-553) and the F domain (hERα amino acids 554-595) was fused to the gene encoding the Gal4 DBD in plasmid pBD-Gal4-Cam to form the “bait plasmid” pBD-Gal-LBD-Cam.

The gene encoding human steroid receptor coactivator-1 (SRC-1) was fused to the Gal4 AD in plasmid pGAD424 to form “prey plasmid” pGAD424SRC-1. Both plasmids were transformed and expressed in S. cerevisiae YRG-2, which contains a Gal4-regulated HIS3 reporter construct integrated on its chromosome. In the presence of agonistic ligands, the LBD undergoes a conformational change and binds to SRC-1, which brings the Gal4 DBD and the Gal4 AD in proximity, thus activating the transcription of the reporter gene. A simple diagram is presented in Figure 3.3 (A) showing the two hybrid system in yeast. The cell growth rate in medium lacking histidine is proportional to the strength of ligand-receptor interaction. We have validated this system using the yeast cells with WT LBD in 96-well plates where cell density (A600) was measured. We could show (Figure 3.3 B) that the cells bearing pBD-Gal4-LBD-Cam and pGAD424-SRC-1 responded to E2 at sub-nanomolar concentration and the EC50 value was 5X10^-10 M. No growth was observed in 96-wells where only solvent (EtOH) was added instead of E2.

A. B.

1.00E-11 1.00E-10 1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 Concentration (Log M)

OD600

Figure 3.3: A. Diagram showing the classical two hybrid system that was used in YRG2 yeast strain. The LBD and SRC-1 were encoded by two different plasmids where they have in their amino terminal end Gal4 DBD and Gal4 AD, respectively. Ligand induced conformational change, dimerization and nuclear transport enable the chimera to bind to the Gal UAS of reporter gene HIS3, enabling nutritional complementation in selection plate. B. Transactivation profile in the yeast two hybrid cells for the WT LBD construct in presence of E2 (blue triangle) or solvent (pink

concentration dependent manner.